Bioactive pentapeptides from rice bran and use thereof

ABSTRACT

In general, the invention relates to novel bioactive pentapeptides from heat stabilized defatted rice bran having anti-cancer, anti-obesity, anti-Alzheimer and other health-promoting activities proteins. The bioactive pentapeptides can be incorporated into pharmaceutical, nutraceuticals and food compositions having at least the bioactive pentapeptide as an active ingredient.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.No. 61/287,517, “Protein Hydrolysates and Peptides with AnticancerActivities from Rice Bran and Methods of Preparation Thereof,” filedDec. 17, 2009, which is incorporated in its entirety herein byreference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to bioactive proteins, peptides andenzymatic hydrolysates from rice bran and use thereof, and moreparticularly to a purified, unmodified and modified bioactivepentapeptide from heat stabilized defatted rice bran having anti-cancer,anti-obesity, anti-Alzheimer and other health-promoting activities. Thebioactive pentapeptides can be incorporated as an active ingredient intopharmaceutical, nutraceutical and food compositions.

2. Description of the Related Art

The treatment of cancer, obesity-related and age-related chronicdiseases are life-long and can have side effects. Americans aresupplementing traditional health care by turning to nutraceuticals andfunctional foods. Nutraceuticals are considered safe, effective andingestible for a lifetime without toxicity, reversibly inhibiting typeand, also amenable to clinical trials. These, when proven effective, canfunction as alternatives to high-cost prescriptions, and could be thenext generation natural anti-disease agents delivered at low cost andhigh efficacy. The use of nutraceuticals in foods, beverages andsupplements is well established in Japan and is gaining interest in theUnited States.

In the United States, cancer is the second most leading cause of death.The Centers for Disease Control and Prevention (CDC) estimated nearly1.4 million new cases of cancer in 2008 and half a million deaths due tocancer occurred in 2007. Colorectal cancer is the second leading causeof cancer-related death in the United States. Over the past decade, theincidence of colorectal cancer has not decreased, and hence there hasbeen no improvement in the mortality rate. Treatment and preventiveoptions for colorectal cancer mainly focus on early detection. There isevidence that dietary components are one of the most importantenvironmental factors in the cause of the colorectal cancer and hencemay act as suitable markers or dietary determinants, which when modifiedand prepared in a biofunctional form can serve as bioactive compounds inreducing the incidence of colorectal cancers. Liver cancer is the mostcommon cancer type in developing countries, but is less common (2% ofcancer deaths) in United States, affecting twice as many men as women.The American Cancer Society has estimated 21,370 new cases in 2008 with18,410 liver-cancer-related deaths in the United States in 2007.Chemotherapy, radiotherapy and liver transplantation are the treatmentoptions available for treating liver cancer. As alternative treatment,to reduce the risk of developing cancer, the focus is on identifyingcompounds present in natural foods that could bear anti-liver cancerproperties.

In addition to cancer, the risk of chronic complications fromobesity-related diseases is a leading cause of death in the UnitedStates. The growing epidemics of type-2 diabetes and cardiovasculardisease are linked to obesity, and in fact, nearly 90% of diabetics arecaused by obesity. Consequently, obesity has triggered impaired glucosetolerance in nearly 197 million people worldwide. Estimated cases ofdiabetes in the United States currently stand at 19.2 million comprising30-40% of world's type-2 diabetics. Roughly 63.1% of Americans have beenidentified to be obese with a basal metabolic index (BMI) of at least25.0. In particular, childhood obesity in the United States has tripledin the past two (2) decades.

Following the ranks of cancer and cardiovascular disease and theirrelated complications in causing death, is a neurological disorder,Alzheimer's disease. It has been thought that blood vessel damage in thebrain, most likely to occur in patients with diabetes and highcholesterol, can lead to symptoms of Alzheimer's disease and, preventingthese states can reduce the risk of developing Alzheimer's disease. One(1) in eight (8) persons aged 65-85 and nearly half of persons over age85 have Alzheimer's disease. In 2011, baby boomers (those born between1946 and 1964) will begin turning 65, reaching the age that stratifiesgreatest risk for Alzheimer's disease. To reduce the risk forAlzheimer's disease, many have turned to alternative treatments based oncompounds present in natural foods that could bear anti-Alzheimer'sdisease properties.

Several bioactive components in nutraceuticals have demonstratedanti-oxidant, anti-obesity, anti-angiogenic and anti-hypertensiveactivities and hypocholesterolemic and immunomodulatory effects.Peptides and proteins from food sources can aid in cancer prevention andtreatment. For example, whey proteins and α-lactalbumin have been shownto inhibit colon cell proliferation. Cereal grains and their componentsare widely investigated for the presence of bioactive components to beused in nutraceuticals. Cereal grains are known to possess high-qualityprotein, which when consumed, are broken down by gastrointestinal (GI)proteolytic enzymes to release bioactive peptides. Cereal grainsincluding rice, wheat and legumes, including soybeans, and theirrespective components, have been investigated for the presence ofbioactive proteins and peptides. For example, Oryzatensin, anileum-contracting bioactive peptide obtained from rice albumin, has beenshown to have an immuno-stimulatory role. Similarly, proteolytichydrolysis of soybean protein using Alcalase and Proteinase S enzymesresulted in peptides that were anti-hypertensive and anti-oxidative,respectively. Rice and its components have also been studied to exertspecific anti-disease properties, such as anti-oxidative,anti-carcinogenic and anti-mutagenic. Constituents such as proteins andpeptides from rice or co-products of rice milling, however, have onlybeen studied to a limited extent.

Enzymatic hydrolysis has been one of the main approaches to producebioactive peptides from soybean, wheat, corn, rice, barley, buckwheatand sunflower proteins. The potential bioactive nature of components ofrice, however, is not well known, including the bran portion, which isnutritionally beneficial but used as a low cost animal feed. It is alsoimportant that the peptides be resistant to GI environment when ingestedfor being metabolically bioactive.

Rice bran is a cheap co-product of rough rice milling having nutrientsincluding B vitamins, minerals, and fiber, including oil, which hashealth benefits. It is being used as a low-cost animal feed and thestate of Arkansas contributes over 50% of the overall rice production inthe United States. Defatted rice bran has approximately 20% protein,with the proteins and their peptide fragments being complexed withincarbohydrates and lipids providing difficulties in protein extraction.

It is therefore desirable to provide bioactive proteins, peptides andenzymatic hydrolysates from rice bran and use thereof.

It is further desirable to provide a purified, unmodified and modifiedbioactive pentapeptide from heat stabilized defatted rice bran havinganti-cancer, anti-obesity, anti-Alzheimer and other health-promotingactivities.

It is yet further desirable to provide bioactive pentapeptides that canbe incorporated as an active ingredient into pharmaceutical,nutraceutical and food compositions.

It is still further desirable to provide bioactive pentapeptides havingunique sequences that renders its bioactive nature, which can enhanceglucose uptake into cells, act to sequester bad cholesterol and fattyacids accumulating in excessive BMI states, or even mitigate molecularevents taking place in neuronal cells during the onset of Alzheimer'sdisease.

SUMMARY OF THE INVENTION

In general, in a first aspect, the invention relates to a bioactivepentapeptide comprising the amino acid sequence Glu-Gln-Arg-Pro-Arg (SEQID NO: 1). The bioactive pentapeptide may be isolated from heatstabilized defatted rice bran or non-defatted rice bran, and exhibitsanti-cancer, anti-obesity and/or anti-Alzheimer activity. Theanti-cancer activity of the bioactive pentapeptide may be an inhibitoryactivity on proliferation of human colon, liver, breast and/or lungcancer cell lines. The bioactive pentapeptide may be modified,including: the side chain glutamine group being glycosylated, methylatedor modified by deamidation; the side chain glutamic acid group and theside chain glutamine group being modified to form a pyroglutamateacid-peptide; and at least one side chain arginine group being modifiedwith a food grade dicarbonyl substance.

In general, in a second aspect, the invention relates to apharmaceutical composition comprising the bioactive pentapeptide and apharmaceutically acceptable carrier. The pharmaceutical composition maybe for topical administration as a lotion, gel or an emulsion or fororal administration as a dietary supplement or as a food ingredient.Also, the pharmaceutical composition may include a derivative or analogof the bioactive pentapeptide.

In general, in a third aspect, the invention relates to a food productcomprising the bioactive pentapeptide of SEQ ID NO: 1 and a foodsubstance. The food substance may take the form of: beverages, such asnon-alcoholic and alcoholic drinks, soft drinks, sport drinks, energydrinks, fruit juices, lemonades, teas and milk-based drinks; dairyproducts, such as yogurts; and fortified foods, such as bakery items andas snacks, cereal-based foods and breakfast cereals.

In general, in a fourth aspect, the invention relates to a method oftreatment or prevention of diseases, such as cancer, obesity orAlzheimer's. The method includes the steps of administering atherapeutically effective amount of a composition comprising thebioactive pentapeptide. The method of treatment or prevention of cancermay be for proliferation of human colon, liver, breast and/or lungcancer. The composition of the method may be administered topically as alotion, gel or an emulsion or administered orally as a dietarysupplement or as a food ingredient. In addition, the composition can bea pharmaceutical, nutraceutical or food composition.

In general, in a fifth aspect, the invention relates to the use of atleast one bioactive pentapeptide comprising the amino acid sequenceGlu-Gln-Arg-Pro-Arg (SEQ ID NO: 1) in the manufacture of a nutraceuticalcomposition.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a Box-Behnken RSM 3 points prediction profiler selection ofthe optimum conditions needed for proteolytic digestion in accordancewith an illustrative embodiment of the bioactive pentapeptides from ricebran and use thereof disclosed herein;

FIG. 2 is a graphical representation of the viability of Caco-2 cellsafter exposure to rice bran peptide fractions measured by trypan bluedye exclusion assay;

FIG. 3 is a graphical representation of the viability of HepG2 cellsexposed to rice bran peptide fractions measured by trypan blue dyeexclusion assay;

FIG. 4 is a graphical representation of a MTS assay for thedetermination of the anti-cancer activities of GI-resistant, rice branpeptide fractions on Caco-2 and HepG2 cells;

FIG. 5 is a graphical representation of a MTS assay for thedetermination of the anti-cancer activities of the bioactivepentapeptides from rice bran peptide <5 kDa fractions (GI-resistantfractions) on HCT-116 cells;

FIG. 6 is a graphical representation of a MTS assay for thedetermination of the anti-cancer activities of the bioactivepentapeptides from rice bran peptide <5 kDa fractions (GI-resistantfractions) on HTB-26 cells;

FIG. 7 is a graphical representation of a clonogenic toxicity assay ofthe bioactive pentapeptides from rice bran peptide <5 kDa fractions(GI-resistant fractions) on HCT-116 cells;

FIG. 8 is a process flow chart illustrating an example of the stepsinvolved towards the preparation of rice bran peptide fractions inaccordance with an illustrative embodiment of the bioactivepentapeptides from rice bran and use thereof disclosed herein;

FIG. 9 is a graphical representation of a MTS assay for thedetermination of the anti-cancer activities of ion exchangechromatography (IEC) eluted fractions of the bioactive pentapeptidesfrom rice bran disclosed herein;

FIG. 10 is a HPLC profile of 50 mM IEC eluate from IEC. Peaks werecollected every 10 min of the run and analyzed for anti-cancer activity.The 60-70 min peak (arrowhead) showed anti-cancer activity, which wasfurther purified;

FIG. 11 is a HPLC profile of a purified, single peptide from 60-70 minfraction from IEC, and the 62 min peak (arrowhead) was collected forevaluation of anti-cancer effect;

FIG. 12 is a graphical representation of a MTS assay for thedetermination of the anti-cancer activities of a purified bioactivepentapeptide from rice bran peptide <5 kDa fractions in accordance withan illustrative embodiment of the bioactive pentapeptides from rice branand use thereof disclosed herein;

FIG. 13 is a graphical representation of a dose response at 24 h of thepurified bioactive pentapeptide on various lines of human cancer cells;

FIG. 14 is a MALDI profile showing molecular weight of the purifiedbioactive pentapeptide obtained after HPLC purification;

FIG. 15 is a tandem MS-MS PSD fragmentation of the purified bioactivepentapeptide of FIG. 14;

FIG. 16 is a graphical representation of cell viability of adipocytesupon treatment with the purified bioactive pentapeptide from rice branpeptide <5 kDa fractions. The values are means of three (3) replicationswith standard deviations, and the cell viability was measured using thetrypan blue dye exclusion assay;

FIG. 17 is a graphical representation of cell viability of amyloidpeptide-induced neuronal cells upon treatment with the purifiedbioactive pentapeptide. The values are means of three (3) replicationswith standard deviations, and the cell viability was measured using theMTS assay. The control represents amyloid-induced neuronal cells withoutpeptide treatment;

FIG. 18 is a graphical representation of the degree of glycosylationobserved with increasing time at varying relative humidity (RH)fractions in accordance with an illustrative embodiment of the bioactivepentapeptides from rice bran and use thereof disclosed herein.Peptide-glucose mixture samples were drawn every 12 h to determinedegree of glycosylation at varying RH. Plotted values are mean oftriplicate analysis;

FIGS. 19 a, 19 b and 19 c are graphical representations of theevaluation of enhanced inhibition of glycos-peptides prepared at varyingconditions in accordance with an illustrative embodiment of thebioactive pentapeptides from rice bran and use thereof disclosed herein.In FIG. 19 a, the glycos-peptides were prepared at varying RH at pH 6;in FIG. 19 b, the glycos-peptides were prepared at varying RH at pH 7;and in FIG. 19 c, the glycos-peptides were prepared at varying RH at pH8. Peptide-glucose mixture samples were maintained at 45, 55, and 65 RHprepared at pH 6, 7 and 8 for 24 h. Plotted values are means withstandard deviations;

FIG. 20 is a graphical representation of evaluation of enhancedinhibition of glycos-peptide prepared at varying temperatures at pH 7,RH 55. Peptide-glucose mixture samples were prepared at RH 55, pH 7 at40, 50 and 60° C. temperatures and tested for growth inhibition activityon colon cancer cells. Plotted values are means with standarddeviations; and

FIG. 21 illustrates an example of the structure of the bioactivepentapeptide isolated and characterized from rice bran having multiplesite anti-proliferative activity against cancers, obesity andAlzheimer's disease in accordance with an illustrative embodiment of thebioactive pentapeptides from rice bran and use thereof disclosed herein.

Other advantages and features will be apparent from the followingdescription and from the claims.

DETAILED DESCRIPTION OF THE INVENTION

The compositions and methods discussed herein are merely illustrative ofspecific manners in which to make and use this invention and are not tobe interpreted as limiting in scope.

While the compositions and methods have been described with a certaindegree of particularity, it is to be noted that many variations andmodifications may be made without departing from the spirit and scope ofthis disclosure. It is understood that the compositions and methods arenot limited to the embodiments set forth herein for purposes ofexemplification.

In general, the invention relates to bioactive proteins, peptides andenzymatic hydrolysates from rice bran and use thereof, and moreparticularly to a purified, unmodified and modified bioactivepentapeptide from heat stabilized defatted rice bran having anti-cancer,anti-obesity, anti-Alzheimer and other health-promoting activities.Additionally, the bioactive pentapeptide has a unique sequence thatrenders its bioactive nature, which can enhance glucose uptake intocells, act to sequester bad cholesterol and fatty acids accumulating inexcessive BMI states and/or mitigate molecular events taking place inneuronal cells during the onset of Alzheimer's disease. In addition, thebioactive pentapeptide may be an effective anti-hypertensive,anti-mutagenic and anti-microbial agent.

The bioactive pentapeptide refers to a peptide having the amino acidsequence of Glu-Gln-Arg-Pro-Arg (EQRPR) (SEQ ID NO: 1) or astructural/functional variant thereof. The C-terminus end of thebioactive pentapeptide includes three (3) amino acids, Arg-Pro-Arg, withGlu-Gln amino acids in the N-terminus end. The bioactive pentapeptide ofSEQ ID NO: 1 has an exact mass of 685.378 Da. The presence of charged(glutamic acid) and heterocyclic amino acid (proline) in the sequence ofthe bioactive pentapeptide may attribute to the health-promotingproperties. A variant to the bioactive pentapeptide could be made byintroducing mutations in SEQ ID NO: 1 at positions not essential to thehealth-promoting activities. Further, amino acid substitutions ofreplacing one amino acid residue with another having similar chemicalproperties is unlikely to affect the activity of a peptide, and suchmutations is also expected to preserve the health-promoting activitiesof any structural variants of the bioactive pentapeptide. Thehealth-promoting activities of the bioactive pentapeptide can bedetermined by assays described in the following examples. Furthermore,the bioactive pentapeptide can be isolated from a natural source, suchas rice bran, produced by the expression of a recombinant nucleic acidmolecule, or can be chemically synthesized.

A method for making the bioactive pentapeptide includes the: (1)preparation using food grade materials including alkali/acid and/orproteolytic enzymes and fermentation; and (2) separation of proteinhydrolysates from heat stabilized defatted rice bran (HDRB) ornon-defatted rice bran followed by generation of GI-resistant peptides,which are fractionated to obtain definite molecular sized fractions ofthe bioactive pentapeptide. When the rice bran is defatted and directlyenzymatically hydrolyzing the high-quality protein using endoprotease,the method of making the bioactive pentapeptide disclosed hereinsustainably releases peptides in a consistent manner.

Due to its excellent health-promoting activities in vitro, the bioactivepentapeptide, or functional/structural variant thereof, can beincorporated as an active ingredient into pharmaceutical, nutraceuticaland food compositions for preventing or treating various cancer lines,obesity-related diseases and/or Alzheimer's disease, as well as actingas an anti-hypertensive, anti-mutagenic and/or anti-microbial agent.These compositions incorporating the bioactive pentapeptide may furthercontain protective hydrocolloids, such as gums, proteins, modifiedstarches, binders, film forming agents, encapsulating agents/materials,wall/shell materials, matrix compounds, coatings, emulsifiers, foamingagents, surface active agents, solubilizing agents, e.g., oils, fats,waxes, lecithins etc., adsorbents, carriers, fillers, co-compounds,dispersing agents, wetting agents, processing aids (solvents), flowingagents, flavoring agents, sweetening agents, coloring agents, weightingagents, jellyfying agents, gel forming agents, anti-oxidants,anti-microbial and other preservative agents.

Moreover, a multi-vitamin and mineral supplement may be added to thecompositions incorporating the bioactive pentapeptide to obtain anadequate amount of an essential nutrient, which is missing in somediets. The multi-vitamin and mineral supplement may also be useful fordisease prevention and protection against nutritional losses anddeficiencies due to lifestyle patterns. In addition, the compositionshaving the bioactive pentapeptide may be incorporated into beverages,e.g., non-alcoholic and alcoholic drinks, soft drinks, sport drinks,energy drinks, fruit juices, lemonades, teas and milk-based drinks,along with other dairy products and/or fortified food and bakery goods.

Further, the pharmaceutical, nutraceutical and food compositions may bein any galenic formulation that is suitable for administrating to thehuman body, especially in any form that is conventional for oraladministration, e.g., in solid form such as (additives/supplements for)food or feed, food or feed premix, fortified food or feed, tablets,pills, granules, capsules, and effervescent formulations, such aspowders and tablets, or in liquid form, such as solutions, emulsions orsuspensions, e.g., beverages, pastes and oily suspensions. The pastesmay be filled into hard or soft shell capsules, whereby the capsulesfeature, e.g., a matrix of (fish, swine, poultry, cow) gelatin, plantproteins or ligninsulfonate. Examples for other acceptable forms ofadministration are transdermal, parenteral and injectable. Thepharmaceutical, nutraceutical and food compositions may be in the formof controlled immediate or sustained release formulations.

The bioactive pentapeptide provides a method for prophylactic ortherapeutic treatment of various cancer lines in a patient byadministering to the patient the bioactive pentapeptide, or afunctional/structural variant thereof, at an amount effective forproviding anti-cancer activity. In addition, the bioactive pentapeptideprovides a method of prophylactic or therapeutic treatment of obesityand obesity-related diseases in a patient by administering to thepatient the bioactive pentapeptide, or a functional/structural variantthereof, at an amount effective for providing anti-obesity activity.Moreover, the bioactive pentapeptide provides a method of prophylacticor therapeutic treatment of Alzheimer's in a patient by administering tothe patient the bioactive pentapeptide, or a functional/structuralvariant thereof, at an amount effective for providing anti-Alzheimer'sdisease activity. It is understood that the actual amount of thebioactive pentapeptide to be administered can vary in accordance withthe age, size, condition and other factors associated with the specificpatient to be treated, depending upon the discretion of medicalprofessionals.

The bioactive pentapeptide from rice bran and use thereof disclosedherein is further illustrated by the following examples, which areprovided for the purpose of demonstration rather than limitation.

Materials and Methods Isolation of Protein Hydrolysates HavingAnti-Cancer Properties from Heat-Stabilized Defatted Rice Bran

Rice bran, an economical, under-utilized co-product of rough ricemilling was used to produce peptide hydrolysates, which wereinvestigated for various health-promoting activities. Proteinhydrolysates prepared by Alcalase hydrolysis under optimized conditionswere treated further to obtain gastrointestinal resistant peptidehydrolysates, which were fractionated into >50 kDa, 10-50 kDa, 5-10 kDaand <5 kDa sizes, and evaluated for inhibitory activity on proliferationof human colon (Caco-2; HCT-116), liver (HepG2), breast (MCF-7; HTB-26)and lung (NCI-H1299) epithelial cancer cell lines by trypan blue dyeexclusion assay. By way of example, GI resistant <5 kDa and 5-10 kDasized peptide fractions inhibited growth of Caco-2 cells by 80% and <5kDa fraction inhibited growth of HepG2 cells by ˜50% compared tocontrols and non-resistant fractions. An MTS cell titer assay confirmedanti-proliferative effects of the peptide fractions. The resultsdemonstrated that 5-10 kDa and <5 kDa sized GI resistant fractionspromoted significant (p<0.05) inhibitory activities on cancer cell linescompared to controls.

Materials. HDRB was obtained from Riceland Foods (Stuttgart, Ark.), theRomicon ultrafiltration system from Koch Membrane Systems(Massachusetts, USA), and food-grade Alcalase enzyme from a bacterialstrain purchased from Novozyme (North Carolina, USA). Human colon(Caco-2; HCT-116), liver (HepG-2), breast (HTB-26) and lung (NCI-H1299)epithelial cancer cell lines were purchased from ATCC (Manassas, Va.).Dulbecco's modified Eagle's medium, fetal bovine serum and gentamycinwere purchased from Hyclone (Logan, Utah). MTS kits were purchased fromPromega (Madison, Wis.). Hp 1090 series, HPLC system, reverse-phase C-18peptide column, analytical grade, sephadex G-75 resin from PharmaciaBiotech AB (Uppsala, Sweden), biopore C18 preparative HPLC column, aminoacid analyzer from Beckman Coulter, Bruker Reflex III (Bruker DaltonicsGMBH, Bremen, Germany) and Bruker Ultraflex II time-of-flight massspectrometers at the Statewide Mass Spectrometry Facility, University ofArkansas. All other chemicals purchased were of HPLC grade and purchasedfrom Sigma-Aldrich Corp. (St. Louis, Mo.).

Enzymatic Hydrolysis of HDRB to Obtain Protein Hydrolysates:Approximately 500 g of ground and sieved HDRB (passed through a 60 mesh)was dissolved and homogenized with 0.6 L deionized water and stirred for30 min at room temperature. Based on a response surface methodoptimization design, HDRB was digested with 3.5 AU (Anson units ofenzyme) Alcalase (4970 μL) at pH 8.0 and heated at 50° C. for 60 min. Toarrest proteolytic digestion, the enzyme was inactivated by heating thesuspension at 85° C. for 3 min. The hydrolysis mixture was centrifugedat 3,000 g for 15 min to obtain the hydrolysates in the supernatant. Thesupernatant was freeze-dried and stored at 4° C. until needed.

Degree of Hydrolysis. The degree of hydrolysis was determined accordingto the OPA method using serine as standard. The sample solution wasprepared by dissolving approximately 0.1 g to approximately 1.0 g offreeze-dried hydrolysate in 100 mL of deionized water. Serinestandard/sample solution (400 μL) was added to the test tube (time 0)containing 3 mL of OPA reagent, mixed for 5 s, and allowed to stand for2 min, and then the absorbance was read at 340 nm in thespectrophotometer.

The degree of hydrolysis was calculated as follows: DH=h/h_(tot)×100%,where h is the number of cleaved peptide bonds and h_(tot) is the totalnumber of peptide bonds per protein calculated ash_(tot)=(serine-NH₂-0.4), where serine-NH₂ is the meqv of serine NH₂ pergram of protein.

${{Serine}\text{-}{NH}_{2}} = \frac{\begin{matrix}\begin{matrix}{\left( {{OD}_{sample} - {OD}_{blank}} \right) \times 0.9516\mspace{14mu} {{meqv}/L} \times} \\{\left( {{sample}\mspace{14mu} {volume}\mspace{14mu} {in}\mspace{14mu} L} \right) \times}\end{matrix} \\{100/\left( {{sample}\mspace{14mu} {wt}\mspace{14mu} {in}\mspace{14mu} g \times {protein}\mspace{14mu} \%} \right)}\end{matrix}}{\left( {{OD}_{standard} - {OD}_{blank}} \right)}$

Protein Content. Protein contents were determined according to theKjeldahl method. A Kjeldahl 2006 Digester (Foss Tecator, Hoganas,Sweden) was used to digest samples.

Generating GI Juices Resistant Bran Hydrolysates. It is essential thatthe peptides be resistant to the GI tract to impart uninhibitedbiological function and bioavailability. Hence, the bran hydrolysateswere passed through a simulated gastric and intestinal solution asdescribed below.

Simulated gastric juice was prepared as follows: To deionized water (90mL) in a 100 mL volumetric flask were added sodium chloride (0.2 g) andconcentrated hydrochloric acid (0.7 mL) and stirred for 30 min. Thefinal volume was made up to 100 mL with deionized water and transferredinto a beaker. The pH was adjusted to 2.0. Purified enzyme pepsin (0.32g) was added and stirred. The temperature of the solution was maintainedat 37° C. Five grams (5 g) of freeze-dried hydrolysate was dissolved inthe simulated gastric juice and allowed to incubate at 37° C. withconstant shaking. After 120 min, the pH was adjusted to 7.2 toinactivate the enzyme. The reaction mixture was centrifuged at 3000 gfor 20 min to obtain soluble peptide hydrolysates in the supernatant. Inthe in vitro digestibility studies, the samples were typically examinedup to 120 min. The resistant supernatant hydrolysate was freeze-driedand stored at 4° C.

Simulated intestinal juice was prepared as follows: To deionized water(90 mL) in a 100 mL volumetric flask were added potassium phosphatemonobasic (0.68 g) and sodium hydroxide 0.2 N (7.7 mL) and stirred for30 min. Final volume was made up to 100 mL and transferred into abeaker. The pH of the solution was adjusted to 8.0, and the mixture wasmaintained at 37° C. Pancreatin at a final concentration of 0.1% wasadded and stirred. The simulated gastric juice treated hydrolysate (infreeze-dried form) was dissolved in the simulated intestinal juice andallowed to incubate at 37° C. with constant shaking. After 120 min, theenzyme was inactivated by heating at 85° C. for 10 min. The reactionmixture was then centrifuged at 3000 g for 20 min to obtain solublepeptide hydrolysate in the supernatant. The hydrolysate was stored at 4°C.

Fractionation of GI-Resistant Peptide Hydrolysates by Ultrafiltration.Fractionation was carried out with a Romicon ultrafiltration systemequipped with 1 in. diameter, hollow-fiber polysulfone membranecartridges. Approximately 500 mL of filtered soluble GI-resistantpeptide hydrolysate were run through sequential ultrafiltration columnswith membrane cartridges having nominal molecular weight cutoffs (MWCO)of 50, 10, and 5 kDa. Approximately one third of the volume of thehydrolysate was ultrafiltered through each membrane as retentates andthe rest as permeates. In particular, in each MWCO cartridge, thepeptide hydrolysate was ultrafiltered at a dilution factor of 5.Immediately after the first ultrafiltration, the retentate wasdiafiltered twice with 2 volumes of deionized water. The permeates ofthe first (50 kDa) ultrafiltration and the second diafiltration stepwere pooled and subjected to the second run through the 10 kDa and thenthe 5 kDa MWCO's, respectively. The resulting retentates from each ofthe MWCO were freeze-dried and stored at 4° C. Only GI-resistant peptidehydrolysates were subjected to fractionation and only of those being <5kDa were tested for inhibitory activity against human colon, liver, lungand breast cancer cell lines.

Human Cancer Cell Cultures. Human colon epithelial cancer cell lineCaco-2, liver epithelial cancer cell line HepG2 and breast epithelialcancer cell line HTB-26 were cultured separately at 37° C. in Dulbecco'sModified Eagles Medium (DMEM) in the presence of 10% fetal bovine serum,supplemented with 1 mM L-glutamine, sodium pyruvate, 1 mM sodiumbicarbonate, and 50 μg/mL gentamycin. Human lung epithelial cancer cellline NCI-H1299 was cultured in RPMI-1640 medium supplemented with 10%fetal bovine serum. After cell growth reached 70%, cell viability wasmonitored by employing the trypan blue dye exclusion assay after peptidetreatments. Briefly, the monolayer was allowed to grow for 2-3 days at37° C. on 24-well flat-bottom plates. One hundred microliters (100 μL)of rice bran peptide fractions at 1 mg/mL protein content was added tothe cultures. For positive control genistein at 200 and 400 μM in salineand for negative control saline alone was used. After 24-48 h oftreatments, the medium was removed, and the cells were brieflydissociated with 0.1% trypsin and 0.53 mM ethylenediaminetetraaceticacid (EDTA) solution. Following this, 0.5% trypan blue dye mixed ingrowth medium was added to each well. Samples were then aspirated fromeach well and loaded onto chambers in a hemocytometer cell, and cellcounts were taken. This assay reflected the number of viable cells thatsurvived after treatment with peptide samples.

Human colon cancer cell line HCT-116 was grown in McCoy's 5a mediumsupplemented with 10% fetal bovine serum and 100 μg/mL of penicillin andstreptomycin (GIBCO-BRL). Human breast (HTB-26) cancer cell line wascultured separately at 37° C. in DMEM in the presence of 10% fetalbovine serum, supplemented with 1 mM L-glutamine, sodium pyruvate, 1 mMsodium bicarbonate and 50 μg/mL gentamycin. Cells were grown at 37° C.under a humidified atmosphere of 5% carbon dioxide. Based on the resultsfrom the Caco-2 and HepG2 studies, the <5 kDa peptide fraction was usedbecause of its ability to inhibit growth of cancer cells. Hence <5 kDapeptide fraction was selected for testing against HCT-116 and HTB-26cell lines. After cell growth achieved 70%, they were treated withdifferent concentrations of bran peptide fraction <5 kDa for differenttime periods and evaluated for growth inhibition properties.

MTS and MTT Assay. Cell proliferation inhibition was determined usingthe phenazine methosulfate 3-(4,5-dimethyl thiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTS) mix-based cell titer assay and the[3-(4,5-dimethyl thiazole-2-yl)]-2,5-diphenyl tetrazolium bromide(MTT)-based cell titer assay. After about 36 h of Caco-2, HepG2,HCT-116, HTB-26 and NCI-H1299 cell growth, respectively, cells weretrypsinized, loaded onto a hemocytometer, and counted.

Approximately 1000 cells per well was used for growth onto 96-wellfiat-bottom plates. The cells were allowed to attach and grow for 36 h.After 36 h, old medium was replaced with fresh medium, and samples ofrice bran peptide fractions were treated with the cells for determiningthe effect on growth as a concentration- and time-dependent manner.After 2-4 h of exposure of the rice bran peptides to the monolayer, theMTS mix was added at a final volume of 20 μL/100 μL of medium and thenincubated for an additional 60 min under the same conditions. MTT dyewas added after each time-point followed by the termination of theformation of colored formazan product by a detergent solution. Thereaction was terminated by adding 10% SDS, and the absorbance offormazan was measured at 490 and 570 nm in a Tunable Versamax MicroplateReader (Molecular Devices, Sunnyvale, Calif.). Positive and negativecontrols were used similarly as used in the trypan blue dye exclusionassay, including appropriate row or column of wells was left untreatedat each time-point. All assays were performed in duplicates, and theresults are expressed as mean values ±standard error.

Clonogenic Assay. To determine the anti-cancer activity of rice branpeptides, a clonogenic cytotoxicity assay was performed to test theefficacy of rice bran peptides on proliferating cancer cells. Theclonogenic assay was performed using the HCT-116 cell line. Cells weretrypsinized and a single cell suspension was prepared. Cells were platedat a density of 100 cells per 35 mm well. Cells were treated withdifferent concentrations of the rice bran peptide fraction. After 72 h,the medium was replaced with fresh medium. Cells were allowed to growfor another 8 days and then stained with 0.025% crystal violet. Theexcess crystal violet was removed with 30% methanol, plates were airdried at room temperature, and numbers of colonies were counted.

Data Analysis. Experimental data from the Caco-2 and HepG2 cell cultureswere analyzed using JMP 7.0 statistical software with the leastsignificant differences between samples being P<0.05, and experimentaldata from the HCT-116, HTB-26 and NCI-H1299 cell growths were analyzedusing SigmaPlot statistical software (Systat Software, Inc., San Jose,Calif.) with sample means and standard errors of approximately 10%.Response surface method was used as a model to optimize enzymatichydrolysis of rice bran using Alcalase enzyme.

Example 1 Enzymatic Hydrolysis of Rice Bran by Alcalase Enzyme andProtein Contents

A four-factorial response surface design optimization with optimumdegree of hydrolysis as well as digested protein contents as responsevalues were determined. The four parameters, enzyme concentrations [1.5,3.5 and 5 Alcalase units (AU)], pH (6, 8, and 10), temperature (40, 50,and 60° C.), and incubation time for digestion (30, 60, and 90 min),were fitted to generate optimum concentrations of enzyme, pH, time andtemperature for achieving optimum degree of hydrolysis. Box-Behnkensurface response using the JMP 7.0 statistical software was used toevaluate the interactions between parameters to generate optimum valuesfor enzymatic hydrolysis. A degree of hydrolysis at 23.4% was consideredto be optimum, with an E/S ratio of 0.01.

Digested protein contents (in mg/mL) were obtained for each factorialcombination. For example, at 1.5 AU enzyme concentration, pH 8, and 40°C. for 60 min of digestion the digested protein content was found to be0.993 mg. The prediction profile from the response surface design shownin FIG. 1 enabled selection of the optimum conditions needed forproteolytic digestion. The prediction profile designated 3.5 AU enzymeconcentration at pH 8.0, 50° C., and 60 min time of digestion forobtaining the optimum value for digested protein content, 1.025±0.36mg/mL, with a DH of 23.4%. For consistent production of peptides fromrice bran, optimized conditions were used for performing enzymatichydrolysis.

Example 2 Colon and Liver Anti-Cancer Activity Evaluation ofGI-Resistant and Nonresistant Rice Bran Peptide Hydrolysate Fractions

The trypan blue dye exclusion assay was conducted to determine cellviability after treatment of cells with peptide fractions. This assayevaluates the number of viable cells that remain after exposure ofpeptides on to Caco-2 as well as HepG2 cells. Both GI resistant peptidesas well as non-GI resistant peptides were tested on colon and livercancer cell lines.

FIG. 2 depicts the effect of GI and non-GI resistant peptide fractionson Caco-2 cells. On Caco-2 cells, GI-resistant peptide fractions ofsizes <5 and 5-10 kDa were found to significantly inhibit theproliferation of viable cells compared to higher molecular weightfractions (>10 and >50 kDa), non-GI resistant fractions and negativecontrol. There was an approximately 3-fold reduction in viable cellsbetween GI-resistant and non-GI resistant fractions that were below 10kDa. Similar patterns of inhibition between fractions that were above 10kDa were not observed. The positive control used was genistein atconcentrations of 200 and 400 μM. Genistein is an isoflavone that is aknown anticancer agent. At 200 μM concentration, there were 20000 viablecells/mL on Caco-2 cell line, which was significantly less than thenegative control (lacking genistein) that resulted in over 100000 viablecells/mL.

FIG. 3 depicts the effect of GI- and non-GI-resistant peptide fractionson HepG-2 cells. On HepG-2 cells, GI-resistant peptide fraction <5 kDawas alone shown to inhibit proliferation of viable cells significantlycompared to non-GI-resistant fraction and negative control. Fromexperimental data, it was found that the resistant fractions were morebioactive than the nonresistant fractions. When the peptide fractionswere tested for GI resistance, resistant peptides were generated, whichnot only meant that they were exposed to highly specific enzymaticcleavage, causing them to expose more side chains, but also may implytheir suitability in the digestive tract, rendering absorptive and henceconsumable properties. In the human body, peptides are usually generatedwhen proteins pass through the intestine, where gastrointestinal enzymesact and release the peptides, before absorption. Depending upon thenature of the proteins, peptides, and their amino acid sequences, theseproteins/peptides may exert specific biological functions. Bioactivepeptides when ingested should pass through the intestinal barrier and betransported to the target organs to impart anti-hypertensive,anti-cancer or other health-promoting activities. Thus, gastrointestinalresistant peptide fractions (<5 and 5-10 kDa) tend to open theirbioactive groups, imparting more bioactivity by effectively inhibitingproliferation of both colon and liver cancer cells more than thenonresistant fractions. Moreover, only soluble peptides were generatedin a process of eliminating organic or other constituents of rice branthat could possibly interfere with bioactivity. While rice bran hasorganic bioactive components, soluble peptides derived from enzymatichydrolysis of protein hydrolysates can aid in inhibitory action of humananti-colon and liver cancer cell proliferation and can have truebiological activity in terms of bioavailability and delivery.

To confirm the findings of peptide bioactivity, a more specific assay,the MTS-based titer assay, was conducted. FIG. 4 depicts MTS-based celltiter assay results for confirming inhibitory actions of peptidefractions on colon and liver cancer cells, respectively. This assayreflects cytotoxicity as an indication of early damage to cells therebyreducing metabolic (mitochondrial) activity. There is >80% cytotoxicityto HepG2 cells with the <5 kDa fraction and nearly 70% cytotoxicity toCaco-2 cells. The 5-10 kDa fraction caused 60% cytotoxicity to HepG2cells and 50% cytotoxicity to Caco-2 cells. These results confirm thatresistant peptide fractions <5 and 5-10 kDa inhibit growth of colon andliver cancer cells more effectively than the nonresistant fractions. Forfractions that were >10 kDa pronounced bioactivity was not observed,possibly because the fractions are longer in length requiring more timeof proteolytic exposure to unfold. Since the trypan blue dye exclusionassay was enumerative of the cell viability after peptide treatmentsshowing significant bioactivities with the GI-resistant fractions, onlyGI-resistant fractions were subjected to the MTS assay.

Example 3 Colon and Breast Ant-Cancer Activity Evaluation ofGI-Resistant and Nonresistant Rice Bran Peptide Hydrolysate Fractions

Treatment with rice bran <5 kDa fraction at 24 and 48 h time-pointsrevealed no inhibition in the growth of HCT-116 colon cancer cellscompared to the untreated cells, whereas at the 72 h time-point therewas a dose- and time-dependent inhibition in the growth of HCT-116 cells(FIG. 5). The growth of HCT-116 cells was reduced by nearly 80% afterthe treatment with 650 g/mL of the <5 kDa fraction at the 72 htime-point. Similar results were obtained with HTB-26 breast cancercells. Treatment with rice bran <5 kDa fraction at 24 and 48 htime-points showed no inhibition compared to the untreated cells. At the72 h time-point, however, there was a significant inhibition in thegrowth of HTB-26 cells in a dose- and time-dependent manner (FIG. 6).Like HCT-116 cells, the HTB-26 cells also showed maximum sensitivity at650 g/mL of <5 kDa fraction treatment with a 65% inhibition in thegrowth at 72 h time-point. As noted above, >50, 10-50 and 5-10 kDafractions revealed no growth inhibitory effect on colon and liver cancercells, however, the growth inhibitory activity for colon and breastcancer cells of rice bran peptide is present in the <5 kDa fraction.

The clonogenic assay also indicates the toxicity of the <5 kDa fractionto HCT-116 cells (FIG. 7). The cytotoxicity of the <5 kDa fraction waspronounced after treating the cells with 500 g/mL and that the IC50 doseof the <5 kDa fraction was 770 g/mL. These results indicate that <5 kDapeptide fraction of the rice bran has a potent anti-tumor activity forcolon cancer cells.

As discussed, the screening for determination of anti-cancer activitywas done employing the MTT assay and subsequently confirmed byclonogenic assay on the anti-cancer fraction. The time- andconcentration-dependent growth inhibition patterns observed with the <5kDa peptide fraction on HCT-116 and HTB-26 reveal better results withHCT-116 cells by the MTT assay. This suggests that the fraction couldhave a better and positive impact on reducing progression of human coloncancer. Further, the clonogenic assay, which also reflects cytotoxicityconfirms that a higher dosage and longer time is needed for the <5 kDafraction to have strong inhibitory or cytotoxicity effect on the growthof especially the colon cancer cells. Since <5 kDa fraction showed abetter inhibition on colon cancer cells compared to liver cancer cells,clonogenic assay of the <5 kDa fraction was done only on colon cancercells. The dosage and time-dependent growth inhibition pattern may implythat the fractions may be a slow-acting. Higher doses may reduce thetime needed for killing the maximum number of cells.

Bioactive Pentapeptide from Heal Stabilized Detailed Rice Bran

Based on the above findings that rice bran peptide fractions have theability to cause growth arrest in colon, breast, lung and liver cancercell types in vitro, the <5 kDa fraction originally separated bypressure-driven membrane based separation (ultrafiltration) that showedsignificant anti-cancer effects was subjected to furthercharacterization to yield pure peptide(s) having similar or enhancedanti-cancer properties.

Purification by IEC followed by reverse phase HPLC was done to obtainsingle pure peptide(s) from the >5 kDa fraction. Fifty millimolar (50mM) sodium chloride eluate from ion exchange column caused approximately75% inhibition to colon and liver cancer cells growth and, 60% and 68%inhibitory activities on lung and breast cancer cells, respectively. Theeluate was purified using HPLC using peptide-specific column. It wasobserved that the 60-70 min peak showed enhanced anti-cancer activity,namely, 84% inhibition on colon, 80% on breast and 84% on liver cancercells. Accurate molecular mass of the pure peptide by MALDI-TOF revealeda mass of 685.378 Da. Amino acid analysis revealed the presence ofGlutamic acid, Proline and Arginine. Tandem mass spectrometry fordetermining the amino acid sequence of the pure peptide(s) was doneusing post-source decay fragmentation analysis. The sequence of thebioactive pentapeptide from the rice bran is Glu-Gln-Arg-Pro-Arg (EQRPR)(SEQ ID NO: 1).

Ion Exchange Chromatography: A sephadex G-75 ion exchange resin waspacked into a glass column and equilibrated with 10 mM phosphate buffer,pH 8.0. Ten milliliters (10 mL) of <5 kDa peptide hydrolysate (˜1 mg/mLprotein concentration) was loaded onto the column at 1 mL/min flow rate.The elution was started by washing the unbound hydrolysate eluted with10 mM phosphate buffer until about 5 bed volumes. After washing, thehydrolysate was eluted using 10 mM phosphate buffer containing 50 mMNaCl followed by elution with 10 mM phosphate buffer containing 100 mMNaCl for a total 5 bed volumes. The eluates were collected, concentratedin an Amicon concentrator with buffer exchange and stored at 4° C. Theanti-cancer activity of the eluates obtained after ion-exchange wasdetermined using the MTS assay described above.

Preparative HPLC Purification of IEC Eluate Showing Anti-CancerActivity: Preparative scale peptide-specific column (Biopore Prep ID22×L 250 mm part #34955) was used to separate peptides from the IECeluates that showed better anti-cancer activity and the absorbance ofthe eluate monitored at 215 nm. The gradient from solvent A (1.2 mlTFA/1000 ml deionized water) to solvent B (0.1% TFA inAcetonitrile:water 50:50) was varied from 100% solvent A to 100% solventB over 80 min at 2 ml/min flow rate monitored at 215 nm. The peaks werecollected and tested for anti-cancer activity, and the peak that showedanti-cancer activity was fully characterized using mass spectrometry andamino acid sequencing.

Amino Acid Analysis: A modified method of AOAC 982.30a (1990) was usedfor hydrolyzing purified peptides. Ten milligrams (10 mg) of peptidesamples were hydrolyzed in 10 mL of 6.0 N HCl under vacuum at 150° C.for 12 h and evaporated under nitrogen at 60° C. Sodium diluent bufferpH 2.2 (1 mL) was added to the dried peptide, filtered and the filtratewas analyzed for amino acids. The peptides were pretreated withperformic acid prior to hydrolysis to preserve cysteine and methionine,while alkali hydrolysis was conducted to determine tryptophan (AOAC982.15, 2000). Amino acid analysis of the filtrate was conducted on anautomated amino acid analyzer (Beckman 6300, Beckman Instruments, Inc.,Palo Alto, Calif.) at a flow rate of 0.67 mL/min (0.44 mL/min for buffersolutions and 0.23 mL/min for ninhydrin solution). Sodium citratebuffers (pH 3.3, 4.3 and 6.3) were used as eluents. The amino acidcontents (in g/100 g sample) were quantified by comparing them withamino acid profiles from external amino acid standards as follows:

(Peak_(sample)/Peak_(standard))×Concentration_(standard)×MW_(standard)

Mass Spectrometry Characterization of Pure Pentapeptide: For preliminaryintact mass determination, 1.0 μl of the HPLC purified pentapeptide wasmixed with 1.0 μl saturated HCCA and spotted on Bruker MTP 384 groundstainless steel MALDI target. Matrix assisted laser desorptionionization time of flight mass spectrometry (MALDI-TOF-MS) were acquiredon a Bruker Reflex III and Bruker Ultraflex II time-of-flight massspectrometers operated in the positive-ion reflectron mode. Highresolution exact mass of the same peptide was obtained by using MALDIlonspec 9.4 T Ion Cyclotron Resonance (MALDI-ICR) mass spectrometer. Forthe exact mass measurements, 2,5 dihydrobenzoic acid (DNB) was used asthe MALDI matrix and followed exactly the same spotting technique.

MALDI Fragmentation: Fragmentation of intact peptide ions were performedto obtain sequence information using MALDI post source decay studies(MALDI-PSD). MALDI-PSD fragmentations of these ions were analyzed usingthe “Lift” mode in Bruker Ultraflex II MALDI-TOF-MS.

Data Analysis and de novo Sequencing: Fragmentation pattern obtained byMALDI-TOF-MS was interpreted using Bruker Biotools software, which usesde novo sequencing algorithm to determine the best sequence for theobserved fragmentation pattern.

Example 4 Anti-Cancer Activity of Eluates

On colon and liver cancer cells, the 50 mM NaCl eluate showed around 75%inhibition by the MTS dye assay. On breast cancer cells, there was 68%inhibition, while on lung cancer cells there was 60% inhibition (FIG.9). While the 50 mM eluate showed cancer cell inhibitions better thanthe 100 mM eluates, the 50 mM eluate possibly had the pool of <5 kDapeptides that contribute towards the anti-cancer effect. The 50 mM IECeluate was subjected to HPLC purification using a peptide-specificcolumn.

FIG. 10 shows the HPLC profile of 50 mM IEC eluates. The peaks werecollected at 10 min intervals and tested for anti-cancer activity. Itwas found that the 60-70 min fraction showed anti-cancer activity andthis fraction was further purified in HPLC. FIG. 11 shows thepurification of a single peptide obtained from 60-70 min fractionisolated from ion exchange chromatography. The peak eluted at 62 min wasevaluated for anti-cancer activity and suggests the presence of bothpolar and non-polar amino acids that make up the peptide.

Example 5 Anti-Cancer Activity of Pure Peptide

The pure peptide showed 84% inhibition on colon cancer cells, 80% onbreast cancer cells and 84% on liver cancer cells, very similar to thepositive (inhibitory) control (genistein) at 400 μg/ml. On breast andliver cancer cells, the pure peptide showed 80% and 85% inhibitionsrespectively, while on lung cancer cells there was 69% inhibition (FIG.12). The results show that the peptide bears strong anti-canceractivities better than the <5 kDa peptide fraction from which it waspurified. The purification of a single peptide from the <5 kDa rice branhydrolysate resulted in identifying the anti-cancer component withinrice bran. When its activity is tested and compared against all cancertypes, enhanced activity of the peptide towards lung cancer cells wasnot demonstrated, however, more specific lung cancer types like smallcell, squamous cell, and non-small cell lung cancer carcinomas mayprovide better inhibition of proliferation.

Example 6 Bioactive Pentapeptide

Amino acid analysis revealed the predominance of three (3) amino acidssuggesting the anti-cancer peptide could be a short peptide. As can beseen below in Table 1, the amino acid analysis of the pure peptideobtained after HPLC purification identified were arginine, proline,glutamic acid and glutamine.

TABLE 1 Amino acids Peptide nmol/ml Glutamic Acid 1.50 Proline 0.99Arginine 2.65

Dose-response pattern of the pure peptide (FIG. 13) revealed that from600 μg/mL to 1000 μg/mL maximum inhibition was achieved on most cancercells even at 24 h period. There is an increase in inhibition pattern asthe dosage increases and seems to plateau off beyond 600 μg/mL. Thedosage for the pure peptide is similar to what was observed with the <5kDa fraction on most cancer cell lines as per the earlier studies exceptthere is a 24 h response for significant anti-cancer activity.

MALDI-TOF-MS analysis was performed on the HPLC purified peptide. FIG.14 shows the MALDI-TOF-MS spectrum of the purified fraction and confirmsthe purity of the fraction. The accuracy of the mass was confirmed byMALDI-ICR where the mass of the ion was measured to 10 ppm accuracy. Bymeans of MALDI-TOF-MS, single protonated molecular ions (M+H⁺) of theintact peptide were located at m/z 685.378 for the bioactivepentapeptide.

Further tandem MS-MS was performed on the predominant peak by isolatingthe ion inside the mass spectrometer to enable post source decayfragmentation to obtain amino acid sequence (FIG. 15). Three (3) aminoacids were identified from the C-terminus end as Arg-Pro-Arg. TheN-terminal amino acids when matched against the database showed a highprediction of Glu-Gln amino acids in the N-terminus end. Table 2 belowillustrates de novo sequencing of peptide matched for sequence based onexact mass of 685.378 Da.

TABLE 2 E Q R P R Glu Gln Arg Pro Arg Ion 1 2 3 4 5 1 2 3 4 5 a E Q R PR 102.055 230.114 386.215 483.267 639.369 b E Q R P R 130.050 258.108414.210 511.262 667.363 y E Q R P R 175.119 272.172 428.273 556.331685.374 i E Q R P R 102.055 101.070 129.113 70.065 129.113 5 4 3 2 1 ArgPro Arg Gln Glu

The de novo sequencing thus revealed the peptide to have the amino acidsequence as Glu-Gln-Arg-Pro-Arg (EQRPR) (SEQ ID NO: 1). The presence ofcharged (glutamic acid) and heterocyclic amino acid (proline) in thesequence could have attributed anti-cancer properties to the peptide.

Anti-Obesity and Anti-Alzheimer's Properties of Bioactive PentapeptideIsolated from Heat Stabilized Defatted Rice Bran

The bioactive pentapeptide also possesses the ability to promotewellness through health benefits by reducing the risk of chroniccomplications including obesity- or age-related diseases, such asAlzheimer's disease. After confirming the anti-cancer properties, thebioactive pentapeptide was again isolated and characterized by fractionsof peptides from rice bran for possible inhibitory effects againstAlzheimer's and obesity. The bioactive pentapeptides were prepared ofprotein/peptide hydrolysates from rice bran by treating the hydrolysateswith simulated GI environments to obtain GI resistant peptidesfractionate the resistant peptides to ultrafiltration to generatepeptide fractions based on molecular size, evaluate the roles ofresistant peptide fractions for bioactivities against obesity andAlzheimer's disease using cell culture models, and finally characterizepeptide(s) using chromatography and mass spectrometry.

Minimal hydrolysis and GI juices treatment followed by fractionationresulted in <5 and 5-10 kDa fractions. With <5 and 5-10 kDa fractionsthe preadipocytes showed differentiation and proliferation (60%)significantly more compared to undifferentiated cells (controls) (25%).Approximately 35% reduction in cytotoxicity of amyloid-inducedneuroblastoma cells that were treated with peptide fractions <5 and 5-10kDa compared to the cytotoxicity was observed with amyloid-induced cells(control) that were not treated with peptides. The bioactivepentapeptide, Glu-Gln-Arg-Pro-Arg (EQRPR) (SEQ ID NO: 1), wascharacterized from the <5 kDa fraction showing bioactive effects. Itshowed nearly 70% adipocyte viability more than control possiblysignifying insulin-like differentiation to confer protective roleagainst obesity. In addition, the bioactive pentapeptide showed nearly a45% reduction in cell cytotoxicity on amyloid-induced neuronal cells.The bioactive pentapeptide provides an efficient and reproduciblebiocatalytic technology that utilizes an underutilized co-product, ricebran, to produce anti-Alzheimer's and anti-obese value-added bioactivepeptide, which can be incorporated into pharmaceutical, nutraceuticaland food compositions having at least the bioactive pentapeptide as anactive ingredient.

Materials: Unless otherwise noted, the materials utilized where the sameas enumerated in the forgoing examples of the bioactive pentapeptide anduse thereof. Human pre-adipocytes and adipocyte basal growth medium anddifferentiation medium (Lonza, USA), Neuroblastoma cell line (IMR-32)and growth medium (ATCC, Manassas, USA), and media supplements includingfetal bovine serum, gentamycin, were purchased from Hyclone (Logan,Utah). Preparative liquid chromatography system LC-8A was purchased fromShimadzu, USA.

Preparation of Rice Bran Peptide Fractions: HDRB was enzymaticallyhydrolyzed with food grade Alcalase under optimum degree of hydrolysisfollowing a response surface design to obtain protein hydrolysates. Thehydrolysates were treated with simulated GI resistant solutions togenerate GI resistant peptides, which were then fractionated into <5,5-10, 10-50, and >50 kDa fractions as fully discussed above.

Evaluation of Degree of Differentiation of Adipocytes for Accumulationof Lipids: Undifferentiated human preadipocytes were allowed todifferentiate upon treatment with a known differentiating agent [0.25μmol/L DEX (IS-IBMX-DEX) mixture]. To determine the role of peptidefractions on adipocyte differentiation, peptide fractions were added tosubstitute for insulin or supplemented to DEX mixture. Microscopicobservation of accumulation of lipids using a phase contrast microscope(Tissue culture facility, University of Arkansas) as well as glucoseuptake if any, were determined to record the degree of differentiationand hence, anti-obese property, if any on the peptide fractions. Atrypan blue dye exclusion assay was conducted to determine the adipocytecell viabilities before and after differentiation upon treatments withthe peptides.

Evaluation of Anti-Alzheimer's Disease Activity: Human neuroblastomacells (IMR-32-ATCC Number CCL-127) was used as model system to evaluatethe protective role of rice bran peptide fractions against amyloid beta(1-42) (Cat. No. PP69, EMD Chemicals, Inc., San Diego, Calif.) dependenttoxicity. Human neuroblastoma cells were grown in the presence of betaamyloid peptide with and without bran peptide fractions at differentlevels. Cell survival to the amyloid-induced neuroblastoma cells uponbran peptide treatments was examined by the MTS[(3-(4,5-dimethylthiazole-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt] assay, and compared to the cytotoxicity observed withamyloid-induced neuroblastoma cells.

Purification and Characterization of the Bioactive Pentapeptide Isolatedfrom the <5 kDa Fraction: As fully discussed above, ion exchangechromatography and reverse-phase HPLC were employed to purify thebioactive pentapeptide fully characterized using mass spectrometry andamino acid sequencing.

Example 7 Anti-Obesity Effect

Obesity results in lipid accumulation and differentiation in adipocytes.Observation of lipid accumulation and rate of differentiation ofadipocytes upon treatment with peptide fractions can determineanti-obese property (non or reduced differentiation of adipocytes) ofpeptide fractions, if any. Visceral preadipocytes were allowed to growin the presence of peptide fractions without the differentiating factor.The degree of differentiation into adipocytes was observed visually, andcell counts were taken by trypan blue dye exclusion assay. With <5 and5-10 kDa fractions, the preadipocytes showed differentiation andproliferation significantly more compared to undifferentiated cells(controls). FIG. 16 shows that nearly 60% cells proliferated better thancontrol (undifferentiated cells (˜25% proliferation)), possiblysignifying differentiation-inducing characteristics of the HDRB peptidefractions

Moreover, the bioactive pentapeptide isolated from the <5 kDa fractionshowed nearly 70% adipocyte viability more than control possiblysignifying insulin-like differentiation to confer obesity protectiverole. Most of the obesity regulating peptides (YY) are GI based, wherethey act like gut hormones (leptin) in conferring protective rolesmainly by improving digestion and nutrient absorption. The significanceof testing peptide fractions through simulated GI tract adds importanceby enabling the peptides to resist GI degradation, an important aspectof all gut-regulating peptides.

Example 8 Anti-Alzheimer's Effect

Neuroblastoma cells were treated with amyloid peptide followed bytreatment with rice bran peptide fractions. Cytotoxicity to theamyloid-induced neuroblastoma cells was examined by the MTS assay. FIG.17 shows the extent of cytotoxicity observed with amyloid-induced cellswith and without peptide treatments.

A nearly 35% reduction was observed in cytotoxicity of amyloid-inducedneuroblastoma cells that were treated with peptide fractions <5 and 5-10kDa compared to the cytotoxicity observed with amyloid-induced cells(control) that were not treated with peptides. The bioactivepentapeptide is able to confer protective role against Alzheimer'spathology, and further the bioactive pentapeptide isolated from ricebran showed nearly 45% reduction in cell cytotoxicity on amyloid-inducedneuronal cells better than the control and the <5 kDa fraction.

Modified Bioactive Pentapeptide from Heat Stabilized Defatted Rice Bran

An RSM design with a 3-factorial model was designed to use combinationsof pH, RH (relative humidity), temperature and degree of glycosylationto evaluate enhanced anti-cancer activity of a modified (glycosylated)peptide. Peptide-glucose mixture samples were prepared at the followingconditions/combinations: RH 55, 65, & 75 at pHs 6, 7 and 8 maintained at40, 50 and 60° C. temperatures and tested for growth inhibition activityon colon cancer cells. The degree of glycosylation increased withincreasing time at varying RH, and achieved a maximum of ˜20% degree ofglycosylation. As fully discussed below, glycosylated-peptide preparedat RH 55 at pH 7 or 8 showed slightly better cancer cell activityinhibition (˜85%) compared to the unmodified bioactive pentapeptide(˜80%). The preparation was achieved at 60° C. The bioactivepentapeptide has one glutamine, and hence, only one (1) site forglycosylation, although only ˜20% of the bioactive pentapeptide wasglycosylated with D-glucose. Other glycosylating agents, such asdextrin, may be utilized to promote increased glycosylation of thebioactive pentapeptide, and hence possibly an enhanced cancer cellgrowth inhibition.

Optimization for Degree of Glycosylation: The bioactive pentapeptide(0.02 g) (SEQ ID NO: 1) and D-glucose (0.002 g) were dissolved in waterto give a 10% (w/v) protein solution. The solution pH was adjusted tovarying pHs based on RSM design (6, 7, 8) and stirred for 10 min, andfreeze-dried. The freeze-dried mixture was placed on an aluminum plate,and incubated at 50° C. at varying relative humidity (45%, 65%, 75%)based on the RSM design illustrated below in Table 3.

TABLE 3 Pattern pH RH Temp Glycos 0−−0 7 45 40 30 +00− 8 60 50 10 −0−0 660 40 30 0−0+ 7 45 50 50 0000 7 60 50 30 0+0− 7 75 50 10 0−0− 7 45 50 1000+− 7 60 60 10 0000 7 60 50 30 +0+0 8 60 60 30 +0−0 8 60 40 30 00++ 760 60 50 0++0 7 75 60 30 −00+ 6 60 50 50 +−00 8 45 50 30 −0+0 6 60 60 30−00− 6 60 50 10 −+00 6 75 50 30 0+−0 7 75 40 30 +00+ 8 60 50 50 0+0+ 775 50 50 00−− 7 60 40 10 0−+0 7 45 60 30 ++00 8 75 50 30 00−+ 7 60 40 500000 7 60 50 30 −−00 6 45 50 30 +0+0 8 60 60 80 −−00 6 45 50 80 0−−0 745 40 80 0−0+ 7 45 50 100 0+−0 7 75 40 80 +−00 8 45 50 80 +00− 8 60 5060 00+− 7 60 60 60 +00+ 8 60 50 100 0000 7 60 50 80 0+0− 7 75 50 60 −00−6 60 50 60 −+00 6 75 50 80 ++00 8 75 50 80 0+0+ 7 75 50 100 −00+ 6 60 50100 00++ 7 60 60 100 0000 7 60 50 80 0−+0 7 45 60 80 +0−0 8 60 40 800000 7 60 50 80 0++0 7 75 60 80 00−− 7 60 40 60 00−+ 7 60 40 100 −0+0 660 60 80 0−0− 7 45 50 60 −0−0 6 60 40 80

The RSM design is a 3-factorial model designed to use combinations ofpH, RH, temperature and degree of glycosylation and evaluate the bestcombination that shows enhanced anti-cancer activity as response.

Degree of Glycosylation Determination: Degree of glycosylation of theglycosylated bioactive pentapeptide was determined using a fluorescamineassay. The freeze dried peptide-glucose mixtures were dispersed into 10mL of deionized water, stirred continuously for 30 min, and filteredthrough a 0.45 μm syringe filter. The filtrate was diluted 20 times, and200 μL of the diluted filtrate was added with 4 mL of borate buffer(0.02 M potassium tetraborate, pH 8.5) and 1 mL of fluorescamine reagent(15 mg in 100 mL acetone), and vortexed. Blank solution was preparedusing the buffer without the bioactive pentapeptide. Fluorescenceintensity was read after 5 min of reaction time using aspectrofluorophotometer (Shimadzu Model RF-1501, Kyoto, Japan) atexcitation and emission wavelengths of 390 and 475, respectively.

The degree of glycosylation was calculated as follows:

Degree of glycosylation (%)=(Ac−Aa)/Ac×100

where Ac was the fluorescence of unmodified protein and Aa was thefluorescence of the glycosylated peptide.

In addition to glycosylation, the bioactive pentapeptide may be subjectto other modifications set forth below. Based on the structure of thebioactive pentapeptide having two (2) arginines, a single glutamine anda single glutamic acid, the side chains of the glutamic acid, glutamine,and arginine may be modified (FIG. 21). Because of the lack of lysineamino acid, certain modifications, such as acylation, succinylation andmyristolylation, may not be achieved. The following additionalmodifications can be done for the bioactive pentapeptide.

The side chains of glutamic acid and glutamine can be modified to formpyroglutamate, especially since the bioactive pentapeptides haveglutamic acid followed by glutamine in the N-terminal region.Pyroglutamate peptides have been implicated to add functional roles, andhave been identified in food protein hydrolysates and are being preparedin industrial scales. Pyroglutamic acid belongs to constituents ofgenerally recognized as safe herbs and plants, and hence can be usefulfor application in food systems. In order for pyroglutamic acidformation, the bioactive pentapeptide solution is maintained at varyingtemperatures in a water bath (30-60° C.) for up to 4 h or autoclaved at121° C. at 15 psi for 30 min. A response surface design would aid in theselection of conditions favorable for pyroglutamate formation.

The side chain of arginine of the bioactive pentapeptide may also bemodified. Dicarbonyls used to modify arginines include 2,3-butanedione,p-hydroxyphenylglyoxal and 1,2-cyclohexanedione. Of these, butanedioneand cyclohexanedione are approved as food grade chemicals. Hence, tomodify the arginine group present in the peptide, butanedione andcyclohexanedione can be used, such as by preparing 10 mM solution of2,3-butanedione/1,2-cyclohexanedione in water and adjust pH toapproximately 9.0. Ten microliters (10 μl) aliquot of the solution isadded to 50 μl aliquots of peptide solution (10 mM), which is thenincubatated for 60-180 min. The modified sample is passed through asepandex-iec column and elute with deionized water. The number ofmodified arginines can be determined by measuring the absorbance of thepurified modified peptide at 340 nm.

In addition, the bioactive pentapeptide may be modified by deamidationof glutamine. Deamidation may be achieved at neutral pH when the peptidesolution (10 mM) is left at 37° C., in 0.15 M Tris HCl for 24-72 h. Themodified bioactive pentapeptide can be examined with time after elutingfrom a sephadex IEC column, and change in mass identified by massspectroscopy. The bioactive pentapeptide could also be modified bymethylation of glutamine. The methylated bioactive pentapeptide could beformed by preparing 1 M iodoacetamide (IAM) or S-adenosyl methionine(SAM) stock solution in deionized water. Then, a 1 M Formaldehyde stocksolution in deionized water is prepared, and 20 μl of 1M IAM or SAMsolution is added followed by 40 μL of 1 M formaldehyde solution to 10μl peptide sample (˜1 mg/ml). The solution is then incubated at 4° C. indark on a gel shaker maintained at 100 rpm for 2 h, at which point asize exclusion chromatography can be performed on themethylated-modified bioactive peptide using 20 m M Tris, 150 mM NaCl, pH8.0 for buffer exchange and elution. The methylated-modified bioactivepeptide using SDS PAGE and mass spectroscopy.

Example 9 Anti-Cancer Activity of Glycosylated Bioactive Pentapeptide

Based on the results illustrated in FIGS. 18 through 20, the modified(glycosylated) bioactive pentapeptide prepared at RH 55 at pH 7 or 8showed slightly better cancer cell activity inhibition (˜85%) comparedto the unmodified bioactive pentapeptide (˜80%). The preparation wasachieved at 60° C. Other temperatures were also tested based on the RSMdesign, and the following results pertain to the modified bioactivepentapeptide prepared at different temperatures at one particular RH andpH that showed better anticancer activity.

Glycosylated proteins and peptides have been shown to improvefunctionality in certain food systems owing to the presence of specificgroups in amino acid side chains that are sites for modification. Theamino acid side chains play a major role in modification reactions, andhave been shown to alter the structural integrity of the protein orpeptide, thus influencing functionality. In general, proteins andpeptides containing amide-rich arginine and glutamine can be subjectedto glycosylation modifications. Again, the glycosylated bioactivepentapeptide has one glutamine and hence was the only site forglycosylation, although only ˜20% of the peptide was glycosylated withD-glucose. There was nearly 5% increase in bioactive (anti-cancer)property of modified (glycosylated) bioactive pentapeptide. At aglycosylating degree of 20%, glucose-peptide mixture prepared at pH 7and 8, maintained at RH 55 and temperature 60° C. showed an increase ofcancer inhibiting property by 5% compared to the unmodified bioactivepentapeptide. Other glycosylating agents, such as dextrin, may beutilized to promote increased glycosylation of the peptide, and hencepossibly an enhanced cancer cell growth inhibition.

Whereas, the compositions and methods have been described in relation tothe drawings and claims, it should be understood that other and furthermodifications, especially to enhance anti-disease and otherhealth-promoting activities and bioactivities of peptide fractions, purepeptides and modified peptides, apart from those shown or suggestedherein, may be made within the spirit and scope of this invention.

1. A bioactive pentapeptide comprising the amino acid sequenceGlu-Gln-Arg-Pro-Arg (SEQ ID NO: 1).
 2. The pentapeptide of claim 1wherein the bioactive pentapeptide is isolated from heat stabilizeddefatted rice bran or non-defatted rice bran.
 3. The pentapeptide ofclaim 1 wherein the bioactive pentapeptide exhibits anti-cancer,anti-obesity and/or anti-Alzheimer activity.
 4. The pentapeptide ofclaim 3 wherein said anti-cancer activity is an inhibitory activity onproliferation of human colon, liver, breast and/or lung cancer celllines.
 5. The pentapeptide of claim 1 wherein the side chain glutaminegroup of the bioactive pentapeptide is glycosylated, methylated ormodified by deamidation.
 6. The pentapeptide of claim 1 wherein the sidechain glutamic acid group and the side chain glutamine group of thebioactive pentapeptide are modified to form a pyroglutamateacid-peptide.
 7. The pentapeptide of claim 1 wherein at least one sidechain arginine group of the bioactive pentapeptide is modified with afood grade dicarbonyl substance.
 8. A pharmaceutical composition,comprising: a bioactive pentapeptide having the amino acid sequenceGlu-Gln-Arg-Pro-Arg (SEQ ID NO: 1); and a pharmaceutically acceptablecarrier.
 9. The pharmaceutical composition of claim 8 wherein thepharmaceutical composition is for topical administration as a lotion,gel or an emulsion or for oral administration as a dietary supplement oras a food ingredient.
 10. The pharmaceutical composition of claim 8further comprising a derivative or analog of the bioactive pentapeptide.11. A food product, comprising: a bioactive pentapeptide having theamino acid sequence Glu-Gln-Arg-Pro-Arg (SEQ ID NO: 1); and a foodsubstance.
 12. The food product of claim 11 wherein the food substanceis in the form of beverages, such as non-alcoholic and alcoholic drinks,soft drinks, sport drinks, energy drinks, fruit juices, lemonades, teasand milk-based drinks, in the form of dairy products, such as yogurts,and in the form of fortified foods, such as bakery items and as snacks,cereal-based foods and breakfast cereals.
 13. A method of treatment orprevention of diseases selected from the group consisting of cancer,obesity and/or Alzheimer's, said method comprising the steps of:administering a therapeutically effective amount of a compositioncomprising a bioactive pentapeptide having the amino acid sequenceGlu-Gln-Arg-Pro-Arg (SEQ ID NO: 1).
 14. The method of claim 13 whereinsaid method of treatment or prevention of cancer is proliferation ofhuman colon, liver, breast and/or lung cancer.
 15. The method of claim13 wherein the composition is administered topically as a lotion, gel oran emulsion or administered orally as a dietary supplement or as a foodingredient.
 16. The method of claim 13 wherein the composition is apharmaceutical, nutraceutical or food composition.
 17. The use of atleast one bioactive pentapeptide comprising the amino acid sequenceGlu-Gln-Arg-Pro-Arg (SEQ ID NO: 1) in the manufacture of a nutraceuticalcomposition.